Alterations in cholesterol metabolism restrict HIV-1 trans infection in nonprogressors

Abstract

ABSTRACT HIV-1-infected nonprogressors (NP) inhibit disease progression for years without antiretroviral therapy. Defining the mechanisms for this resistance to disease progression could be important in determining strategies for controlling HIV-1 infection. Here we show that two types of professional antigen-presenting cells (APC), i.e., dendritic cells (DC) and B lymphocytes, from NP lacked the ability to mediate HIV-1 trans infection of CD4(+) T cells. In contrast, APC from HIV-1-infected progressors (PR) and HIV-1-seronegative donors (SN) were highly effective in mediating HIV-1 trans infection. Direct cis infection of T cells with HIV-1 was comparably efficient among NP, PR, and SN. Lack of HIV-1 trans infection in NP was linked to lower cholesterol levels and an increase in the levels of the reverse cholesterol transporter ABCA1 (ATP-binding cassette transporter A1) in APC but not in T cells. Moreover, trans infection mediated by APC from NP could be restored by reconstitution of cholesterol and by inhibiting ABCA1 by mRNA interference. Importantly, this appears to be an inherited trait, as it was evident in APC obtained from NP prior to their primary HIV-1 infection. The present study demonstrates a new mechanism wherein enhanced lipid metabolism in APC results in remarkable control of HIV-1 trans infection that directly relates to lack of HIV-1 disease progression. IMPORTANCE HIV-1 can be captured by antigen-presenting cells (APC) such as dendritic cells and transferred to CD4 helper T cells, which results in greatly enhanced viral replication by a mechanism termed trans infection. A small percentage of HIV-1-infected persons are able to control disease progression for many years without antiretroviral therapy. In our study, we linked this lack of disease progression to a profound inability of APC from these individuals to trans infect T cells. This effect was due to altered lipid metabolism in their APC, which appears to be an inherited trait. These results provide a basis for therapeutic interventions to control of HIV-1 infection through modulation of cholesterol metabolism.

FIG 1

DC and B cells from NP do not mediate HIV-1 trans infection of autologous or heterologous CD4⁺ T cells. (A to C) APC derived from eight PR, eight NP, and seven SN were loaded with R5 HIV-1 BaL and cocultured with autologous (A and B) or heterologous (C; n = 7 per group) CD4⁺ T cells. Concentrations of HIV-1 p24 in the cell culture supernatants were tested every 4 days for 16 days. Individuals were independently tested multiple times with similar results (see the text). P < 0.03 by two-way ANOVA. (D) CD4⁺ T cells from the subjects tested in the experiments shown in panels A and B were cultured for the indicated times to test endogenous autologous virus production. (E) CD4⁺ T cells from the same subjects as in panel A were infected in cis with 3 or 300 pg/ml of HIV-1 BaL, and p24 concentrations were determined as described above. For clarity, p24 concentrations in cultures loaded with the lower MOI are shown only at the latest time point (day 16). Data represent mean p24 concentrations from triplicate wells ± SE. (F) B cells or DC from four NP (NP1, NP2, NP3, and NP4) and four SN were incubated with HIV-1, and virus capture was measured by p24 ELISA. *, below the limit of detection; ns, no statistically significant difference.

FIG 2

DC and B cells from NP do not have altered surface marker expression. DC (A) and B cells (B) from seven PR, eight NP, and seven SN were stained with MAbs against the indicated cell markers. Bars represent the mean percentages of positive cells ± the SE.

FIG 3

Lack of trans infection is not a consequence of HIV-1 infection. (A) B cells from two NP (NP7, NP8) and two PR (PR5, PR8) collected before (gray bars) and after (diagonal bars) HIV-1 seroconversion (SC) were loaded with HIV-1 and cocultured with autologous CD4⁺ T cells. Supernatants were tested after 12 days of coculture, and p24 levels were measured by ELISA. (B) Autologous CD4⁺ T cells from subjects used in Fig. 2A were loaded with HIV-1 BaL at an MOI of 300 pg of p24/10⁶ cells and cultured in parallel to assess susceptibility to cis infection. *, below the limit of detection.

FIG 4

Alteration of cholesterol levels inhibits B-cell-mediated trans infection of T cells. (A) NR ligand treatment inhibits trans infection. B cells from three SN were either left untreated or treated with the LXR ligand TO-901317 or the PPARγ ligand ciglitazone prior to pulsing with HIV-1. Supernatants were collected at the indicated time points for assessment of p24 concentrations. Each result is the average of three sets of triplicate wells per time point (one set per subject). (B) Statin inhibits trans infection. B cells and DC from two SN were left untreated or treated with lovastatin prior to pulsing with HIV-1. Treated cells and controls were then cocultured with autologous CD4⁺T cells in RPMI supplemented with 10% charcoal-stripped FBS. Supernatants were collected at the indicated time points for measurement of p24 concentrations. Each value is the average of triplicate wells for two SN. (C) Cholesterol depletion inhibits trans infection. B cells from two SN were left untreated or treated with BCD, loaded with HIV-1, and cultured with autologous CD4⁺ T cells. Supernatants were collected at the indicated time points, and p24 concentrations were determined by ELISA. Each result is the average of two sets of triplicate wells from two independent experiments. (D) Cholesterol content in cells left untreated or treated with TO-901317 or BCD. Results of a representative experiment of two independent determinations are shown. *, below the limit of detection.

FIG 5

DC and B cells from NP have low cholesterol content. DC (A), B cells (B), and CD4⁺ T cells (C) obtained from PR (PR1, PR2, PR3, PR7, and PR8), NP (NP1, NP2, NP3, NP6, and NP8), or SN were lysed with 1% Triton X-100; total cholesterol was measured with the Amplex Red assay kit; and results were normalized for total protein content. Differences between groups were compared by using a two-tailed t test. ns, no statistically significant difference.

FIG 6

Cholesterol reconstitution in APC from NP confers on them the ability to trans infect T cells. (A) APC (DC and B cells) and CD4⁺ T cells from four NP (NP1, NP2, NP3, and NP6) were left untreated or incubated in 300 µM cholesterol (Ch) for 1 h at 37°C. Cells were extensively washed, pulsed with HIV-1, and cocultured as shown. APC + Ch, APC treated with cholesterol; CD4 + Ch, CD4⁺ T cells treated with cholesterol. (B) APC (DC and B cells) and CD4⁺ T cells from three SN were left untreated or incubated in 300 µM cholesterol for 1 h at 37°C. Cells were extensively washed and cocultured as shown. APC + Ch, APC treated with cholesterol; CD4 + Ch, CD4⁺ T cells treated with cholesterol. (C) Cholesterol contents of DC, B cells, and CD4⁺ T cells from the four NP were measured before (−) and after (+) in vitro cholesterol reconstitution. *, P < 0.05. (D) Cholesterol contents of DC, B cells, and CD4T cells from two SN were measured before (−) and after (+) in vitro cholesterol reconstitution.

FIG 7

ABCA1 levels and effect of ABCA1 knockdown on HIV-1 trans infection. Levels of ABCA1 mRNA in three NP (NP2, NP3, and NP4) were determined by RT-PCR and compared to those in three PR (PR 2, PR3, and PR7). P values were determined by Student’s t test. (B) ABCA1 knockdown restores trans infection. B cells from two NP (NP2 and NP3) were transfected with siRNA for ABCA1, treated with siRNA off-target controls, or left untreated; loaded with HIV-1; and cocultured with autologous CD4⁺ T cells for up to 12 days, when supernatants were collected and tested for HIV-1 p24 by ELISA. (C) Representative data from three independent experiments showing the mean fluorescence intensity (MFI) for ABCA1 on transfected B cells. (D) Efficiency of transfection was determined with parallel cultures transfected with the pmaxGFP plasmid (Lonza) and analyzed by flow cytometry. Positive cells (black histogram) were gated against the untreated control (gray histogram). GFP, green fluorescent protein.